3. Materials and Methods
Reagents, chemicals, hydrophobic carrier octyl-Sepharose® CL-4B, and lipase from Candida rugosa (CRL) were purchased from Sigma-Aldrich (Milano, Italy). Acetyl xylan esterase (AXE) from Bacillus pumilus was from Dobfar (Tribiano, Italy). SepharoseTM CL-6B (agarose) was from GE Healthcare (Milan, Italy). α-d-Mannose pentaacetate (1) was purchased from Sigma-Aldrich (Milano, Italy).
Compounds purification was performed by flash chromatography using Silica Gel high-purity grade, pore size 60 Å 70–230 mesh, 63–200 μm (Sigma-Aldrich). Analytical thin layer chromatography (TLC) was performed on silica gel F254 precoated aluminium sheets (0.2 mm layer, Merck, Darmstadt, Germany). Products were detected by spraying with 5% H2SO4 in ethanol, followed by heating to ca. 150 °C. Enzymatic reactions and activity assays were monitored by Titrator 718 stat (pH-Stat) Tritino from Metrohm (Herisau, Switzerland). Characterization of purified compounds was performed by NMR spectroscopy. NMR spectra were recorded in CDCl3 on a Bruker Advance III 400 MHz spectrometer (Bruker Corporation, Billerica, MA, USA), available at the Centro Grandi Strumenti of the University of Pavia. All 1D and 2D NMR spectra were acquired using the standard pulse sequences available with Bruker Topspin 3.6 software package. Chemical shifts (δ) are given in ppm and were referenced to the solvent signals (δH 7.28, δC 77.00). Signal multiplicities are abbreviated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; b, broad. Structures assignment was performed by means of 2D-COSY and HSQC and, in some cases, 2D-NOESY. Spectra analyses were carried out using Mestrenova reader software. For compounds 5a, 5b, 14, and 18 high resolution mass spectra (HRMS) were recorded with a Bruker Micro-TOF spectrometer in electrospray ionization (ESI) mode, using Tuning-Mix as reference. For all other compounds, mass spectra were recorded on an LCQ-DECA Thermo Finnigan Spectrometer by the ESI (Electron Spray Ionization) ionization method with an ionic source and with use of Xcalibur 2.2 software (Thermo-Finnigan, San Jose, CA, USA). Analyses were run under positive modality, and the experimental conditions were: voltage of the source 5.0 kV, voltage of the capillary 14 V, flow of the gas 35 (arbitrary units), and temperature 200 °C.
3.1. Determination of Enzymatic Activity
The activity of the enzymes was determined following a standard protocol by using an automatic titrator pH-Stat. The hydrolytic activity was calculated based on NaOH consumption (mL of NaOH/min).
3.1.1. Standard Activity Assay with Acetyl Xylan Esterase from Bacillus pumilus (AXE)
The activity of AXE was determined using 1-naphtyl acetate as standard substrate [
21]. The standard reaction mixture was composed of 2 mL of acetonitrile, 2 mL of 1-naphtyl acetate (50 mM in acetonitrile), and 16 mL of phosphate buffer (25 mM, pH 7.0). The reaction was started through the addition of 100 μL soluble enzyme (49 mg/mL) or 10–15 mg of immobilized enzyme. The mixture was mechanically stirred and pH was maintained at 7.0 using 100 mM NaOH as titrant. Experiments were done at least in duplicate.
3.1.2. Standard Activity Assay with Candida rugosa Lipase (CRL)
The activity of CRL was determined using tripropionin as standard substrate [
36]. The standard reaction mixture was composed of 0.6 mL of acetonitrile, 1 mL of tripropionin, and 18.4 mL of Tris-HCl (25 mM, pH 7.0). The reaction was started through the addition of 100 μL soluble enzyme (10 mg/mL) or 10–15 mg of immobilized enzyme. The mixture was mechanically stirred and pH was maintained at 7.0 using 100 mM NaOH as titrant. Experiments were done at least in duplicate.
3.2. AXE Immobilization on Glyoxyl-Agarose (GLX-AG)
GLX-AG was prepared as reported in literature [
37]. Briefly, Sepharose
TM CL-6B (agarose, 5 g) was suspended in deionized H
2O (1.4 mL) and NaOH (1.7 M, 2.4 mL) containing NaBH
4 (28.4 mg/mL). Subsequently, glycidol (1.7 mL) was added dropwise, keeping the vessel at 4 °C in an ice bath. The reaction was kept under gently stirring overnight at 25 °C. After the incubation period, the suspension was filtered, and the carrier was washed abundantly with deionized H
2O. Oxidation was initiated by adding NaIO
4 (100 mM, 34 mL). The reaction was carried out for 2 h at room temperature, and then the carrier was filtered under reduced pressure and washed abundantly with deionized H
2O and stored at 4 °C.
Immobilization of AXE on GLX-AG was performed following a standard protocol [
38,
39]. Briefly, glyoxyl-agarose was washed abundantly with NaHCO
3 buffer (50 mM, pH 10) and then filtered under reduced pressure until dryness. Soluble enzyme (50 mg or 150 mg loading of protein per gram of carrier) was solubilized into NaHCO
3 buffer (50 mM, pH 10). Then, the carrier was added, and the suspension was allowed to stir at 25 °C or 4 °C. Finally, NaBH
4 (1 mg for each 100 mg of carrier) was added to the mixture and incubated for 30 min to allow imino bonds reduction. The immobilized enzyme was then filtered, rinsed thoroughly with distilled water, and stored at 4 °C till use.
3.3. AXE Immobilization on Sepabeads EC-EP/M
Immobilization of AXE on Sepabeads EC-EP/M was performed following a standard protocol [
38,
39]. Briefly, Sepabeads EC-EP/M was allowed to hydrate for 1 h in water on a rolling shaker at 25 °C, and then it was filtered under reduced pressure until dryness. Soluble enzyme (150 mg loading of protein per gram of carrier) was solubilized into KH
2PO
4 buffer (1 M, pH 8). Then, the carrier was added and the suspension was allowed to stir for 24 h at 25 °C. Subsequently, the epoxy groups were quenched with 3 M glycine in KH
2PO
4 buffer (1 M, pH 8) for 18 h at 25 °C. The immobilized enzyme was then filtered, rinsed thoroughly with distilled water, and stored at 4 °C till use.
3.4. CRL Immobilization on Octyl-Sepharose® (OC-AG)
The crude extract of CRL (1.5 g; loading 2500 UI per gram of carrier) was suspended in KH2PO4 buffer (25 mM, pH 7.0). The mixture was allowed to stir on the rolling shaker for 30 min. Then, octyl-Sepharose® (3 g), previously conditioned with the same buffer, was added and the suspension was stirred at room temperature overnight. The enzyme derivative was filtered under reduced pressure on a Büchner funnel, rinsed thoroughly with distilled water, and stored at 4 °C till use.
3.5. Chemical Synthesis of Monosaccharides 2–9
Cyanomethyl 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranoside (2)
Cyanomethyl 2,3,4,6-tetra
-O-acetyl-1-thio-α
-d-mannopyranoside (
2) was synthesized as previously reported [
40].
Briefly, 2-
S-(2,3,4,6-tetra
-O-acetyl-α
-d-mannopyranosyl-)-2-thiopseudourea hydrobromide (7.3 g, 0.017 mmol, 1 eq.), sodium meta bisulphite (6.28 g, 0.034 mmol, 2 eq.), and potassium carbonate (2.81 g, 0.0204 mmol, 1.2 eq) were dissolved in acetone/water (50:50, 80 mL). Subsequently, chloroacetonitrile (21.73 mL, 0.34 mmol, 20 eq.) was added, and the reaction was incubated for ~2 h at room temperature. The reaction mixture was monitored by TLC (ethyl acetate/
n-hexane 5:5, Rf = 0.60). Upon completion, 60 mL of ice water were added to the solution, and the mixture was stirred for 45 min. The reaction was extracted with dichloromethane, and the combined organics extracts were washed with brine, filtered, dried over Na
2SO
4, and concentrated in vacuo. The mixture was then crystallized from hot methanol. A white crystalline solid was obtained (3 g, 45%).
1H-NMR was in agreement with that previously reported [
40].
(4-Methylphenyl) 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranoside (3)
(4-Methylphenyl) 2,3,4,6-tetra
-O-acetyl-1-thio-α
-d-mannopyranoside (
3) was synthesized, slightly modifying the protocol reported by Janssens J. et al. [
41].
Briefly, to a mixture of 1,2,3,4,6-penta
-O-acetyl-α
-d-mannopyranose (
1) (3.372 g, 8.6 mmol, 1 eq.) and
p-thiocresol (0.955 g, 7.6 mmol, 1.2 eq.) in dichloromethane (35 mL), boron trifluoride diethyl etherate (700 μL, 5.6 mmol, 1.5 eq.) was added dropwise. The mixture was stirred at room temperature for 48 h, under nitrogen atmosphere. The reaction mixture was monitored by TLC (ethyl acetate/toluene 3:7, Rf = 0.57). The reaction mixture was then diluted with dichloromethane (28 mL) and washed with saturated NaHCO
3 twice and water. The organic layer was dried over MgSO
4. The solvent was removed, and the residue was purified by column chromatography (SiO
2, ethyl acetate/toluene 3:7). The desired product was obtained as a white solid (3.21 g, 85%).
1H-NMR was in agreement with that previously reported [
42].
Ethyl 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranoside (4)
Ethyl 2,3,4,6-tetra
-O-acetyl-1-thio-α
-d-mannopyranoside (
4) was synthesized following a slightly modified protocol reported by Calosso M. et al. [
43].
Briefly, to a solution of 1,2,3,4,6-penta
-O-acetyl-α
-d-mannopyranose (
1) (3.062 g, 7.85 mmol, 1 eq.) in anhydrous dichloromethane, ethanethiol (0.79 mL, 11 mmol, 1.4 eq.) in the presence of 4 Å molecular sieves was added. The reaction was cooled to 0 °C, and BF
3OEt
2 (1.65 mL, 13.345 mmol, 1.7 eq.) was added dropwise. The reaction was monitored by TLC (ethyl acetate/
n-hexane 6:4, Rf = 0.64). After 7 h, the reaction was washed with 40 mL of a saturated solution of NaHCO
3, and the aqueous phase was washed with dichloromethane. The organic phase was dried with Na
2SO
4, filtered and concentrated in vacuo. The reaction crude was purified by flash chromatography (SiO
2, ethyl acetate/
n-hexane 6:4). The desired product was obtained as a white solid (2.15 g, 70%).
1H-NMR was in agreement with that previously reported [
44].
Propargyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (5)
Propargyl 2,3,4,6-tetra
-O-acetyl-α
-d-mannopyranoside (
5) was synthesized following a standard procedure [
45]. Briefly, 1,2,3,4,6-penta
-O-acetyl-α
-d-mannopyranose (
1) (1.043 g, 2.68 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (8 mL) under nitrogen atmosphere in presence of activated molecular sieves. Propargyl alcohol (0.156 mL, 2.68 mmol, 1 eq.) was added. The mixture was cooled to 0 °C, and BF
3OEt
2 (0.661 mL, 5.36 mmol, 2 eq.) was added dropwise. The reaction mixture was allowed to warm up to room temperature and allowed to stir for 5 days. The solution was diluted with dichloromethane, washed with saturated NaHCO
3 then water, dried over Na
2SO
4, and concentrated in vacuo. The reaction was monitored by TLC (ethyl acetate/
n-hexane 5:5, Rf = 0.30). Column chromatography (SiO
2, ethyl acetate/
n-hexane 5:5) gave the desired compound as white solid (2.64 g, 84%).
1H-NMR was in agreement with that previously reported [
45].
Allyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (6)
Allyl 2,3,4,6-tetra
-O-acetyl-α
-d-mannopyranoside (
6) was synthesized modifying slightly the protocol reported by Balcerzak A.K. et al. [
46]. Briefly, 1,2,3,4,6-penta
-O-acetyl-α
-d-mannopyranose (
1) (1 g, 25.6 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (8 mL) under nitrogen atmosphere in presence of activated molecular sieves. Allyl alcohol (0.175 mL, 25.6 mmol, 1 eq.) was added. The mixture was cooled to 0 °C, and BF
3OEt
2 (0.633 mL, 51.2 mmol, 2 eq.) was added dropwise. The reaction mixture was allowed to warm up to room temperature and allowed to stir for 7 days. The solution was diluted with dichloromethane, washed with saturated NaHCO
3 and then water, dried over Na
2SO
4, and concentrated in vacuo. The reaction was monitored by TLC (ethyl acetate/
n-hexane 5:5, Rf = 0.65). Column chromatography (SiO
2, ethyl acetate/
n-hexane 5:5) gave the desired product as a colorless oil (610 mg, 61%).
1H-NMR was in agreement with that previously reported [
47].
(3-Azidopropyl) 2,3,4,6-tetra-O-acetate-α-d-mannopyranoside (7)
(3-Azidopropyl) 2,3,4,6-tetra
-O-acetate-α
-d-mannopyranoside (
7) was synthesized as previously reported [
34]. Briefly, 3-azido-1-propanol (0.31 mL, 3.08 mmol) and BF
3·Et
2O (0.49 mL, 3.87 mmol) were added to a solution of 1,2,3,4,6-penta
-O-acetyl-α
-d-mannopyranose (
1) (1.0 g, 2.54 mmol) in dichloromethane (20 mL) at 0 °C, and the mixture was stirred at room temperature overnight. The reaction was monitored by TLC (ethyl acetate/
n-hexane 5:5, Rf = 0.25). The reaction mixture was quenched by adding dichloromethane (10 mL) and saturated NaHCO
3 to neutralize the remaining BF
3Et
2O. The aqueous layer was extracted by dichloromethane (30 mL), and the combined organic layers were washed with brine and then dried over anhydrous MgSO
4, affording the crude product. The crude product was purified by flash chromatography (SiO
2, ethyl acetate/
n-hexane 1:1) to yield the desired product as colorless oil (0.58 g, 50%).
1H-NMR was in agreement with that previously reported [
48].
Cyanomethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-d-glucopyranoside (8)
Cyanomethyl 2-acetamido-3,4,6-tri
-O-acetyl-2-deoxy-1-thio-α
-d-glucopyranoside (
8) was synthesized as reported by Zheng C. et al. [
15]. Briefly, 1-thiourea-2-acetamido-3,4,6,-tri
-O-acetyl-2-deoxy-α
-d-glucopyranoside (245 mg, 0.554 mmol) was dissolved in 1:1 water:acetone mixture (2.6 mL), and sodium meta bisulphite (0.212 g, 1.115 mmol), potassium carbonate (0.093 g, 0.672 mmol) and chloroacetonitrile (0.712 mL, 20 eq.) were added. The mixture was stirred at room temperature, and reaction was monitored by TLC (dichloromethane/methanol 9:1, Rf = 0.64). Upon completion, 8 mL of ice water were added to the solution that was stirred for 45 min. The reaction was extracted with dichloromethane, and the combined organics extracts were washed with brine and dried over anhydrous Na
2SO
4 and concentrated in vacuo. The reaction crude was purified by flash chromatography (SiO
2, dichloromethane/methanol 95:5). The desired product was obtained as a white solid (230 mg, 98%).
1H-NMR was in agreement with that previously reported [
15].
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranosyl trichloracetimidate (9)
2,3,4,6-Tetra
-O-acetyl-α
-d-mannopyranosyl trichloracetimidate (
9) was synthesized following the procedure reported by Ekholm F.S. et al. [
49]. Briefly, trichloroacetonitrile (4.97 mL, 49.55 mmol, 5 eq.) and 1,8-diazabicyclo-[5,4,0]-7-undecene (DBU, 0.74 mL, 4.95 mmol, 0.5 eq.) were added to a solution of 2,3,4,6-tetra
-O-acetyl
-d-mannopyranose (
1b) (3.45 g, 9.91 mmol, 1 eq.) in anhydrous dichloromethane (30 mL) at 0 °C under nitrogen atmosphere. The mixture was stirred for 3 h at 0 °C and then concentrated in vacuo. The reaction mixture was monitored by TLC (ethyl acetate/
n-hexane 5:5, Rf = 0.8) and purified by flash chromatography (SiO
2, ethyl acetate/
n-hexane 5:5). The desired product was obtained as a yellow sticky solid (3.89 g, 80%).
1H-NMR was in agreement with that previously reported [
49].
3.6. Enzymatic Deprotection of Monosaccharides 1–8
The deacetylated monosaccharides were produced following a general procedure of hydrolysis.
The substrates (10 mM final concentration) were dissolved in acetonitrile (20%–30% v/v depending on substrate solubility) under magnetic stirring, and then phosphate buffer (50 mM, pH 4.0–5.0) was added slowly. The reaction was started through the addition of immobilized CRL and/or AXE, previously conditioned with reaction buffer. The reactions were performed at 25 °C under mechanical stirring; the pH of the solution was maintained constant by automatic titration. Reaction course was monitored by TLC.
After complete consumption of the starting substrate or before an excessive formation of undesired products, the reactions were stopped by enzymatic derivative filtration on Büchner funnel. Acetonitrile was evaporated under reduced pressure, and the solution was brined and extracted with ethyl acetate. The organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The mixture obtained was purified by flash chromatography.
1,2,3,4-Tetra-O-acetyl-α-d-mannopyranose (1a)
1,2,3,4,6-Penta
-O-acetyl-α
-d-mannopyranose (
1) was hydrolyzed to the corresponding 1,2,3,4-tetra
-O-acetyl-α
-d-mannopyranose (
1a) by CRL-OC-AG as reported by Bavaro T. et al. [
17].
1H-NMR was in agreement with that previously reported [
50].
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranose (1b)
1,2,3,4,6-Penta
-O-acetyl-α
-d-mannopyranose (
1) (30 mg, 5mM) was hydrolyzed to the corresponding 2,3,4,6-tetra
-O-acetyl-α
-d-mannopyranose (
1b) by AXE-GLX-AG.
1H-NMR was in agreement with that previously reported [
49].
Cyanomethyl 2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (2a)
Cyanomethyl 2,3,4,6-tetra
-O-acetyl-1-thio-α
-d-mannopyranoside (
2) was hydrolyzed to the corresponding cyanomethyl-2,3,4-tri
-O-acetyl-1-thio-α
-d-mannopyranoside (
2a) by CRL-OC-AG as reported by Bavaro T. et al. [
17].
1H-NMR was in agreement with that previously reported [
17].
Cyanomethyl 3,4,6-tri-O-acetyl-1-thio-α-d-mannopyranoside (2b)
Cyanomethyl 2,3,4,6-tetra
-O-acetyl-1-thio-α
-d-mannopyranoside (
2) was hydrolyzed to the corresponding cyanomethyl 3,4,6-tri
-O-acetyl-1-thio-α
-d-mannopyranoside (
2b) by CRL-OC-AG as reported by Bavaro T. et al. [
17].
1H-NMR was in agreement with that previously reported [
17].
(4-Methylphenyl) 2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (3a)
(4-Methylphenyl) 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranoside (3) was hydrolyzed to the corresponding (4-methylphenyl) 2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (3a) following the general procedure for enzymatic hydrolysis: substrate (1g, 10 mM) was solubilized in 255 mL of phosphate buffer 50 mM pH 4.0 and 30% v/v of acetonitrile. The reaction was started through addition of CRL-OC-AG (7000 UI). The reaction mixture was monitored by TLC (ethyl acetate/n-hexane 6:4) and purified by flash chromatography (SiO2, ethyl acetate/n-hexane 6:4, Rf = 0,56). The desired product was obtained as a white solid. (590 mg, 65%).
1H-NMR (400 MHz, CDCl3): δ 7.38 (d, 2H, J = 8.0 Hz, Ar), 7.12 (d, 2H, J = 8.0 Hz, Ar), 5.50 (s, 1H), 5.42 (s, 1H), 5.30-5.35 (m, 2H), 4.30 (m, 1H), 3.7 (m, 2H), 2.32 (s, 3H, Ph-CH3), 2.13, 2.09, 2.02 (3s, 9H, OAc).
13C-NMR (300 MHz, CDCl3): 170.74, 169.97, 169.82 (COOCH3), 138.49, 132.67, 130.04, 128.83 (Ar), 86.12, 71.74, 70.98, 69.21, 66.62, 61.30, 21.12 (Ph-CH3), 20.87, 20.75, 20.66 (COOCH3).
MS: m/z = 435.13 [M + Na+] (calculated 435.45).
Ethyl 2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (4a)
Ethyl 2,3,4,6-tetra-O-acetyl-1-thio-α-d-mannopyranoside (4) was hydrolyzed to the corresponding ethyl 2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (4a) following the general procedure for enzymatic hydrolysis: substrate (120 mg, 10 mM) was solubilized in 30.6 mL of phosphate buffer 50 mM pH 4.0 and 30% v/v of acetonitrile. The reaction was started through addition of CRL-OC-AG (500 UI). The reaction mixture was monitored by TLC (ethyl acetate/n-hexane 6:4) and purified by flash chromatography (SiO2, ethyl acetate/n-hexane 6:4, Rf = 0.46). The desired product was obtained as a colorless oil (56.8 mg, 53%).
1H-NMR (400 Hz, CDCl3): δ 5.36-5.28 (m, 4H, H-1, H-2, H-3, H-4), 4.16-4.14 (m, 1H, H-5), 3.70- 3.67 (m, 2H, H-6ab), 2.67-2.62 (m, 2H, SCH2CH3), 2.09, 2.02, 1.93 (3s, 9H, OAc), 1.30 (t, 3H, SCH2CH3).
13C-NMR (400 Hz, CDCl3): δ 170.93, 170.07, 169.79 (COOCH3), 82.15 (C-1), 71.27, 71.06, 69.27, 66.69, 61.23 (5C, ring carbon), 25.39 (SCH2CH3), 20.91, 20.74, 20.65 (COOCH3), 14.68 (SCH2CH3).
MS: m/z = 373.10 [M + Na+] (calculated 373.38).
Propargyl 2,3,4-tri-O-acetyl-α-d-mannopyranoside (5a)
Propargyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (5) was hydrolyzed to the corresponding propargyl 2,3,4-tri-O-acetyl-α-d-mannopyranoside (5a) following the general procedure for enzymatic hydrolysis: substrate (500g, 10 mM) was solubilized in 255 mL of phosphate buffer 50 mM pH 4.0 and 30% v/v of acetonitrile. The reaction was started through addition of CRL-OC-AG (7000 UI). The reaction mixture was monitored by TLC (ethyl acetate/n-hexane 6:4, Rf = 0.37) and purified by flash chromatography (SiO2, ethyl acetate/n-hexane 5:5). The desired product was obtained as a white solid (311 mg, 80%).
1H-NMR (400 MHz, CDCl3): δ 5.33 (dd, 1H, J = 3.4, 10.3 Hz, H-3), 5.27-5.16 (m, 2H, H-2, H-4), 4.98 (s, 1H, H-1), 4.21 (d, 2H, J = 2.4 Hz, OCH2C≡CH), 3.78-3.72 (m, 1H, H-5), 3.65 (dd, 1H, J = 2.4, 12.7 Hz, H-6a), 3.56 (dd, 1H, J = 4.23, 12.7 Hz, H-6b), 2.41 (t, 1H, J = 2.4 Hz, C≡CH), 2.17-2.09-2.02 (3s, 9H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 170.76, 169,97, 169.80 (3C, CH3COO), 96.34 (C-1), 78.06 (CH≡C-), 75.50 (CH≡C-), 71.14, 69.42, 68.72, 66.34, 61.16, 54.96(CH≡CCH2), 20.84, 20.72, 20.66 (3C, CH3COO).
HRMS: m/z = 367.0996 [M + Na+] (calculated 367.100).
Propargyl 3,4,6-tri-O-acetyl-α-d-mannopyranoside (5b)
Propargyl 2,3,4,6-tetra-O-acetyl-α-d-mannopyranoside (5) was hydrolyzed to the corresponding propargyl 3,4,6-tri-O-acetyl-α-d-mannopyranoside (5b) following the general procedure for enzymatic hydrolysis: substrate (500 mg, 10 mM) was solubilized in 234 mL of phosphate buffer 50 mM pH 4.0 and 30% v/v of acetonitrile. The reaction was started through addition of immobilized AXE (787.5 UI). The reaction mixture was monitored by TLC (ethyl acetate/n-hexane 6:4, Rf = 0.41) and purified by flash chromatography (SiO2, ethyl acetate/n-hexane 6:4). The desired product was obtained as a white sticky solid (178 mg, 40%).
1H-NMR (300 MHz, CDCl3): δ 5.29 (t, 1H, J = 9.9 Hz, H-4), 5.17 (dd, 1H, J = 9.9, 3.2 Hz, H-3), 5.01 (d, 1H, J = 1.8 Hz, H-1), 4-26-4.15 (m, 3H, H-6a, OCH2C≡CH), 4.06 (d, 1H, J = 2.5 Hz H-6), 4.02 (m, 1H, H-2), 3.90 (m, 1H, H-5), 2.43 (t, 1H, J = 2.5 Hz, C≡CH), 2.02, 2.01, 1.97 (3s, 9H, COCH3).
13C-NMR (300 MHz, CDCl3): δ 170.85, 169.99, 169.89 (3C, CH3COO), 98.16 (C-1), 78.30 (CH≡C-), 75.33 (CH≡C-), 71.53 (C-3), 69.02, 68.83 (C-2 C-5), 66.12 (C-4), 62.35 (C-6), 54.77 (CH≡C-CH2-O), 20.83, 20.73, 20.67 (3C, CH3COO).
HRMS: m/z = 367.100 [M + Na+] (calculated 367.100).
Allyl 2,3,4-tri-O-acetyl-α-d-mannopyranoside (6a)
Allyl 2,3,4,6-tetra
-O-acetyl-α
-d-mannopyranoside (
6) was hydrolyzed to the corresponding allyl 2,3,4-tri
-O-acetyl-α
-d-mannopyranoside (
6a) following the general procedure for enzymatic hydrolysis: substrate (300 mg, 10 mM) was solubilized in 77.3 mL of phosphate buffer 50 mM pH 4.0 and 30%
v/
v of acetonitrile. The reaction was started through addition of CRL-OC-AG (4372.5 UI). The reaction mixture was monitored by TLC (ethyl acetate/
n-hexane 5:5, Rf = 0.4) and purified by flash chromatography (SiO
2, ethyl acetate/
n-hexane 5:5). The desired product was obtained as a colorless oil (214 mg, 80%).
1H-NMR was in agreement with that previously reported [
51].
(3-Azidopropyl) 2,3,4-tri-O-acetate-α-d-mannopyranoside (7a)
(3-Azidopropyl) 2,3,4,6-tetra
-O-acetate-α
-d-mannopyranoside (
7) was hydrolyzed to the corresponding (3-azidopropyl) 2,3,4-tri
-O-acetate-α
-d-mannopyranoside (
7a) by CRL-OC-AG as reported by Li Z. et al. [
34].
1H-NMR was in agreement with those previously reported [
34].
Cyanomethyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-d-glucopyranoside (8a)
Cyanomethyl 2-acetamido-3,4,6-tri
-O-acetyl-β
-d-glucopyranoside (
8) was hydrolyzed to the corresponding 1-thiocyanomethyl-2-acetamido-3,4-di
-O-acetyl-β
-d-glucopyranoside (
8a) by CRL-OC-AG as reported by Zheng C. et al. [
15].
1H-NMR was in agreement with that previously reported [
15].
3.7. Chemical Synthesis of Disaccharides 10–18
2′,3′,4′,6′-Tetra-O-acetyl-α-d-mannopyranosyl-(1→6)-1,2,3,4-tetra-O-acetyl-α-d-mannopyranose (10)
2,3,4,6-Tetra
-O-acetyl-α
-d-mannopyranosyl trichloracetimidate (
9) (3.77 g, 7.662 mmol, 1.6 eq.) and 1,2,3,4-tetra
-O-acetyl-α
-d-mannopyranose (
1a) (1.67 g, 4.798 mmol, 1 eq.) were dissolved in dry dichloromethane (50 mL) in presence of activated molecular sieves and cooled to 0 °C under nitrogen atmosphere. BF
3OEt
2 (591 μL, 4.798 mmol, 1 eq.) was added, and the mixture was stirred at room temperature for 2.5 h. The reaction was quenched with triethylamine (668 μL, 4.798 mmol, 2 eq.), stirred for 5 min, filtered, and concentrated in vacuo. The reaction mixture was monitored by TLC (dichloromethane/methanol 9:1, Rf = 0.89) and purified by flash chromatography (SiO
2, dichloromethane/methanol 9:1). The desired product was obtained as a white solid (2.83 g, 87%).
1H-NMR was in agreement with that previously reported [
52].
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (11)
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-1-thio-
α
-d-mannopyranoside (
11) was synthesized as reported by Bavaro T. et al. [
17].
Briefly, 2,3,4,6-tetra
-O-acetyl-α
-d-mannopyranosyl trichloracetimidate (
9) (226 mg, 0.460 mmol, 2 eq.) and cyanomethyl 3,4,6-tri
-O-acetyl-1-thio-α
-d-mannopyranoside (
2a) (83 mg, 0.230 mmol, 1 eq.) were dissolved in dry dichloromethane (5 mL) in presence of activated molecular sieves and cooled to −70 °C with dry ice under nitrogen atmosphere. BF
3OEt
2 (56.6 μL, 0.460 mmol, 2 eq.) was added, and the mixture was stirred at room temperature for 4 h. The reaction was quenched with triethylamine (64.2 μL, 0.460 mmol, 2 eq.), stirred for 5 min, filtered, and concentrated in vacuo. The reaction mixture was monitored by TLC (ethyl acetate/
n-hexane 5:5, Rf = 0.32) and purified by flash chromatography (SiO
2, ethyl acetate/
n-hexane 5:5). The desired product was obtained as a white solid (125 mg, 79%).
1H-NMR was in agreement with that previously reported [
17].
(4-Methylphenyl)(2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (12)
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranosyl trichloracetimidate (9) (366 mg, 0.743 mmol, 2.5 eq.) and (4-methylphenyl) 2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (3a) (140 mg, 0.298 mmol, 1 eq.) were dissolved in dry dichloromethane (7 mL) in presence of 4 Å molecular sieves and cooled to 0 °C under nitrogen atmosphere. BF3OEt2 (73.5 μL, 0.596 mmol, 2 eq.) was added, and the mixture was stirred at 0 °C for 3.5 h. The reaction was quenched with triethylamine (83.2 μL, 0.596 mmol, 2 eq.), stirred for 5 min, filtered, and concentrated in vacuo. The reaction mixture was monitored by TLC (ethyl acetate/n-hexane 5:5, Rf = 0.28) and purified by flash chromatography (SiO2, ethyl acetate/n-hexane 5:5). The desired product was obtained as a white solid (204 mg, 92%).
1H-NMR (400 MHz, CDCl3): δ 7.30 (d, 2H J = 8.0 Hz, Ar), 7.08 (d, 2H, J = 8.0 Hz, Ar), 5.50 (dd, J = 1.7, 3.2 Hz, 1H, H-3), 5.42–5.20 (m, 6H, H-4, H-2′, H-3′, H-2, H-1, H-4′), 4.84 (d, 1H, J = 1.5 Hz, H-1′), 4.43–4.38 (m, 1H, H-5), 4.21 (dd, 1H, J = 5.0, 12.2 Hz, H-6′a), 3.99 (dd, 1H, J = 12.3, 2.4 Hz, H-6′b), 3.95–3.88 (m, 1H, H-5′), 3.74 (dd, 1H, J = 11.3, 5.0 Hz, H-6a), 3.59 (dd, 1H, J = 11.3, 2.7 Hz, H-6b), 2.25 (s, 3H, CH3-Ar), 2.08, 2.09, 2.02, 2.02, 1.99, 1.95, 1.92 (7s, 21H, COCH3).
13C-NMR (400 MHz, CDCl3): δ 170.60, 170.14, 169.90, 169.87, 169.74, 169.72, 169.56 (7C, CH3COO), 138.34, 132.58, 130.08, 129.08 (Ar), 97.98 (C-1′), 86.17 (C-1), 70.91 (C-3), 70.22 (C-5), 69.44, 69.31, 69.07, 68.62 (C-5′), 66.85 (C-6), 66.65, 66.00, 62.31 (C-6′), 21.13(CH3-Ar), 20.85, 20.78, 20.75, 20.72, 20.72, 20.66, 20.62 (7C, CH3COO).
MS: m/z = 765.18 [M + Na+] (calculated 765.20).
Ethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (13)
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranosyl trichloracetimidate (9) (58.7 mg, 0.119 mmol, 1 eq.) and ethyl 2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (4a) (41.8 mg, 0.119 mmol, 1 eq.) were dissolved in dry dichloromethane (1 mL) in presence of 4 Å molecular sieves and cooled to 0 °C under nitrogen atmosphere. BF3OEt2 (14.7 μL, 0.119 mmol, 1 eq.) was added, and the mixture was stirred at room temperature for 4 h. The reaction was quenched with triethylamine (17 μL, 0.119 mmol, 1 eq.), stirred for 5 min, filtered, and concentrated in vacuo. The reaction mixture was monitored by TLC (dichloromethane/acetone 9:1, Rf = 0.71) and purified by flash chromatography (SiO2, dichloromethane/acetone 9:1). The desired product was obtained as a white solid (28 mg, 37%).
1H-NMR (400 MHz, CDCl3): δ 5.38–5.22 (m, 7H, H-2′, H-3′, H-4′, H-1, H-2, H-3, H-4), 4.85 (d, 1H, J = 1.7 Hz, H-1′), 4.39 (ddd, 1H, J = 9.1, 6.4, 2.3 Hz, H-5), 4.25 (dd, 1H, J = 12.2, 5.4 Hz, H-6′a), 4.16 (dd, 1H, J = 12.2, 2.4 Hz, H-6′b), 4.06 (ddd, 1H, J = 9.5, 5.4, 2.4 Hz, 1H, H-5′), 3.82 (dd, 1H, J = 10.9, 6.4 Hz, H-6a), 3.56 (dd, 1H, J = 10.8, 2.4 Hz, H-6b), 2.74–2.63 (m, 2H, SCH2CH3), 2.18, 2.17, 2.13, 2.08, 2.05, 2.00, 1.99 (7s, 21H, COCH3), 1.34 (t, 3H, J = 7.4, SCH2CH3).
13C-NMR (400 MHz, CDCl3): δ 170.61, 170.05, 169.99, 169.88, 169.77, 169.74, 169.66 (7C, CH3COO), 97.33 (C-1′), 81.47 (C-1), 71.04, 69.56, 69.54 (C-5), 69.41, 68.88, 68.59 (C-5′), 66.81 (C-6), 66.53, 66.05, 62.39 (C-6′), 25.08 (SCH2CH3), 20.86, 20.84, 20.74, 20.74, 20.69, 20.63, 20.63 (7C, CH3COO), 14.57 (SCH2CH3).
MS: m/z = 703.12 [M + Na+] (calculated 703.19).
Propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-α-d-mannopyranoside (14)
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranosyl trichloracetimidate (9) (1.1 g, 2.233 mmol, 2 eq.) and propargyl 2,3,4-tri-O-acetyl-α-d-mannopyranoside (5a) (0.384 g, 1.115 mmol, 1 eq.) were dissolved in dry dichloromethane (35 mL) in presence of 4 Å molecular sieves and cooled to 0 °C under argon atmosphere. BF3OEt2 (137.6 µL, 1.115 mmol, 1 eq.) was added, and the mixture was stirred at 0 °C for 4 h. The reaction was quenched with triethylamine (155 µL, 1.115 mmol, 1 eq.), stirred for 5 min, filtered over Celite, and concentrated in vacuo. The reaction mixture was monitored by TLC (dichloromethane/acetone 9:1, Rf = 0.61). Column chromatography (SiO2, dichloromethane/acetone 9:1) gave the desired product as a white solid (0.676 g, 90%).
1H-NMR (400 MHz, CDCl3): δ 5.38-5.25 (m, 6H, H-4′, H-3′, H-2′, H-4, H-3, H-2), 5.03 (d, 1H, J = 1.8 Hz, H-1), 4.87 (d, 1H, J = 1.7 Hz, H-1′), 4.32 (d, 2H, J = 2.4 Hz, OCH2C≡CH), 4.28 (dd, 1H, J = 5.3 Hz, 12.4 Hz, H-6′a), 4.15 (dd, 1H, J = 2.4 Hz, 12.2 Hz, H-6′b), 4.12-3.98 (m, 2H, H-5′, H-5), 3.80 (dd, 1H, J = 5.7 Hz, 11 Hz, H-6b), 3.60 (dd, 1H, J = 2.6 Hz, 11 Hz, H-6a), 2.53 (t, 1H, J = 2.4 Hz, C≡CH), 2.18, 2.17, 2.12, 2.07, 2.06, 2.01, 2.00 (7s, 21H, COCH3).
13C-NMR (600 MHz, CDCl3): δ 170.59, 170.04, 169.94, 169.83, 169.79, 169.74, 169.70 (7C, CH3COO), 97.51 (C-1′), 96.03 (C-1), 78.05 (OCH2C≡CH), 75.60 (OCH2C≡CH), 69.85(C-5), 69.35, 69.29, 69.01, 68.98, 68.65 (C-5′), 66.64 (C-6), 66.44, 65.97, 62.42 (C-6′), 54.95 (OCH2C≡CH), 20.84, 20.75, 20.72, 20.70, 20.70, 20.64, 20.62 (7C, CH3COO).
HRMS: m/z = 697.1943 [M + Na+] (calculated 697.1950).
Allyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-α-d-mannopyranoside (15)
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranosyl trichloracetimidate (9) (226.4 mg, 0.460 mmol, 1 eq.) and allyl 2,3,4-tri-O-acetyl-α-d-mannopyranoside (6a) (159 mg, 0.460 mmol, 1 eq.) were dissolved in dry dichloromethane (5 mL) in presence of activated molecular sieves and cooled to 0 °C under nitrogen atmosphere. BF3OEt2 (56 μL, 0.460 mmol, 1 eq.) was added, and the mixture was stirred at 0 °C for 2 h. The reaction was quenched with triethylamine (64.2 μL, 0.460 mmol, 1 eq.), stirred for 5 min, filtered, and concentrated in vacuo. The reaction mixture was monitored by TLC (dichloromethane/acetone 9:1, Rf = 0.74) and purified by flash chromatography (SiO2, toluene/methanol 9:1). The desired product was obtained as white solid (156 mg, 50%).
1H-NMR (400 MHz, CDCl3): δ 5.98-5.88 (m, 1H, CH2CH=CH2), 5.41-5.23 (m, 8H, H-2, H-3, H-4 H-2′, H-3′, H-4′, CH2CH=CH2), 4.88 (d, 1H, J = 1.8 Hz, H-1), 4.86 (d, 1H, J = 1.8 Hz, H-1′), 4.30-4.21 (m, 2H, H-6′a, CH2CH=CH2), 4.16 (dd, 1H, J = 12.2, 2.4 Hz, H-5), 4.14-3.97 (m, 3H, H-5′, H-6′b, CH2CH=CH2), 3.80 (dd, 1H, J = 10.9, 6.0 Hz, H-6a), 3.58 (dd, 1H, J = 10.9, 2.5 Hz, H-6b), 2.17, 2.17, 2.13, 2.07, 2.06, 2.01, 2.00 (7s, 21H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 170.63, 170.18, 169.99, 169.89, 169.89, 169.77, 169.69 (7C, COO), 133.04 (CH2CH=CH2), 118.42 (CH2CH=CH2), 97.43 (C-1), 96.26 (C-1′), 69.61, 69.42, 69.36, 69.17, 68.95, 68.64, 68.55 (CH2CH=CH2), 66.63, 66.63, 66.04 (C-6), 62.42 (C-6′), 20.87, 20.82, 20.74, 20.74, 20.70, 20.70, 20.64 (7C, CH3COO).
MS: m/z = 699.25 [M + Na+] (calculated 699.21).
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-d-glucopyranoside (16)
Cyanomethyl (2′,3′,4′,6′-tetra
-O-acetyl-α
-d-mannopyranosyl)-(1→6)-2-acetamido-3,4-di
-O-acetyl-2-deoxy-1-thio-β
-d-glucopyranoside (
16) was synthesized as reported by Zheng C. et al. [
15]. Briefly, 2,3,4,6-tetra
-O-acetyl-α
-d-mannopyranosyl trichloracetimidate (
9) (235 mg, 0.478 mmol, 2 eq.) and cyanomethyl 2-acetamido-3,4-di
-O-acetyl-2-deoxy-1-thio-β
-d-glucopyranoside (
8a) (86 mg, 0.239 mmol, 1 eq.) were dissolved in dry dichloromethane (25 mL) in presence of activated molecular sieves and cooled to 0 °C under nitrogen atmosphere. BF
3OEt
2 (59 μL, 0.478 mmol, 2 eq.) was added, and the mixture was stirred at room temperature for 2.5 h. The reaction was quenched with triethylamine (67 μL, 0.478 mmol, 2 eq.), stirred for 5 min, filtered, and concentrated in vacuo. The reaction mixture was monitored by TLC (ethyl acetate/diethyl ether 3:2, Rf = 0.39) and purified by flash chromatography (SiO
2, ethyl acetate/diethyl ether 3:2). The desired product was obtained as a as a colorless oil (122 mg, 74%).
1H-NMR (400 MHz, CDCl3): δ 5.95 (d, 1H, J = 9.2 Hz, NH), 5.39–5.17 (m, 4H, H-4′, H-3′, H-2′, H-3), 5.05 (t, 1H, J = 9.3 Hz, H-4), 4.86 (s, 1H, H-1′), 4.80 (d, 1H, J = 10.3 Hz, H-1), 4.29 (dd, 1H, J = 12.4, 5.1 Hz, H-6′a), 4.25-4.09 (m, 2H, H-2, H-6′b), 4.02 (m, broad, 1H, H-5′), 3.83-3.76 (m, 2H, H-6a, H-5), 3.73 (d, 1H, J = 17.1 Hz, CHCN) 3.63-3.54 (m, 1H, H-6b), 3.38 (d, 1H, J = 17.1 Hz, CHCN), 2.17, 2.13, 2.08, 2.07, 2.06, 2.01, 1.99 (21H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 171.22, 170.68, 170.53, 170.09, 169.90, 169.72, 169.42 (7C, CH3COO), 116.40 (SCH2CN), 97.36 (C-1′), 83.30 (C-1), 76.85 (C-5), 73.23 (C-3), 69.38 (C-3′), 68.96 (C-5′), 68.86 (C-2′), 68.83 (C-4), 66.70 (C-6), 66.03 (C-4′), 62.41 (C-6′), 52.76 (C-2), 23.14 (CH3, NHAc), 20.87, 20.79, 20.72, 20.67, 20.65, 20.61 (6C, CH3COO), 14.62 (SCH2CN).
MS: m/z = 713.10 [M + Na+] (calculated 713.18).
Cyanomethyl (2′,3′,4′,6′-Tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-1-thio-α-d-mannopyranoside (17)
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranosyl trichloracetimidate (9) (202 mg, 0.41 mmol, 1 eq.) and cyanomethyl 3,4,6-tri-O-acetyl-1-thio-α-d-mannopyranoside (2b) (148 mg, 0.41 mmol, 1 eq.) were dissolved in dry dichloromethane (5 mL) in presence of activated molecular sieves and cooled to −63 °C under argon atmosphere. BF3OEt2 (50.6 μL, 0.41 mmol, 1 eq.) was added, and the mixture was stirred for 30 min. After, the solution was allowed to warm to room temperature and stirred for 1h. The reaction was quenched with triethylamine (57 μL, 0.41 mmol, 1 eq.), stirred for 5 min, filtered over Celite, and concentrated in vacuo. The reaction mixture was monitored by TLC (dichloromethane/acetone 9:1, Rf = 0.42). Column chromatography (SiO2, dichloromethane/acetone 9:1) gave the desired product as white solid (149 mg, 52.6%).
1H-NMR (400 MHz, CDCl3): δ 5.63 (d, 1H, J = 1.9 Hz, H-1), 5.45-5.39 (m, 2H, H-4, H-3′), 5.31 (t, 1H, J = 9.9 Hz, H-4′), 5.27 (dd, 1H, J = 3.4, 1.9, H-2′), 5.16 (dd, 1H, J = 9.6, 3.3, H-3), 4.96 (d, 1H, J = 1.9 Hz, H-1′), 4.37-4.25 (m, 3H, H-6a, H-6b, H-5), 4.23-4.12 (m, 4H, H-2, H-5′, H-6′a, H-6′b), 3.51 (d, 1H, J =17.1 Hz, SCH2CN), 3.40 (d, 1H, J = 17.1 Hz, SCH2CN), 2.17, 2.17, 2.13, 2.12, 2.08, 2.07, 2.04 (7s, 21H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 170.74, 170.65, 170.32, 169.87, 169.74, 169.51, 169.25 (7C, CH3COO), 115.75 (SCH2CN), 99.15 (C-1′), 83.33 (C-1), 76.70 (C-2), 70.35 (C-3), 70.04 (C-5), 69.60, 69.60 (C-2′, C-5′), 68.30, 66.48, 65.95 (C-4, C-4′, C-3′), 62.60 (C-6′), 61.77 (C-6), 20.84, 20.76, 20.68, 20.64, 20.64, 20.62, 20.62 (7C, CH3COO), 15.91 (SCH2CN).
MS: m/z = 714.21 [M + Na+] (calculated 714.65).
Propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-α-d-mannopyranoside (18)
2,3,4,6-Tetra-O-acetyl-α-d-mannopyranosyl trichloracetimidate (9) (212 mg, 0.43 mmol, 1 eq.) and propargyl 3,4,6-tri-O-acetyl-α-d-mannopyranoside (5b) (150 mg, 0.43 mmol, 1 eq.) were dissolved in dry dichloromethane (8 mL) in presence of activated molecular sieves. The solution was cooled to −50 °C under argon atmosphere. BF3OEt2 (49.7 µL, 0.43 mmol, 1 eq.) was added to the flask, and the solution was allowed to warm to room temperature and stirred for 18 h. The reaction was quenched with triethylamine (155 µL, 1.115 mmol, 1 eq.), stirred for 5 min, filtered over Celite, and concentrated in vacuo. The reaction mixture was monitored by TLC (dichloromethane/acetone 9:1, Rf = 0.70). Column chromatography (SiO2, dichloromethane/acetone 9:1) gave the desired product as a white solid (232 mg, 80%).
1H-NMR (400 MHz, CDCl3): δ 5.45-5.27 (m, 5H, H-3′, H-4, H-4′, H-3, H-2′), 5.18 (d, 1H, J =2.0 Hz, H-1), 4.95 (d, 1H, J = 2.0 Hz, H-1′), 4.29 (d, 2H, J = 2.4 Hz, OCH2C≡CH), 4.28-4.22 (m, 2H, H-6a, H-6b,), 4.20-4.12 (m, 3H, H-5′, H-6′a, H-6′b), 4.07(dd, 1H, J =1.2, 2.0 Hz H-2), 4.00-3.95 (m, 1H, H-5), 2.51 (t, 1H, J = 2.4 Hz, C≡CH), 2.17, 2.16, 2.11, 2.10, 2.06, 2.05, 2.03 (7s, 21H, CH3COO).
13C-NMR (101 MHz, CDCl3): δ 170.86, 170.54, 170.33, 169.83, 169.70, 169.44, 169.33 (7C, CH3COO), 99.20 (C-1′), 96.92 (C-1), 78.11 (OCH2C≡CH), 76.75 (C-2), 75.54 (OCH2C≡CH), 70.06 (C-2′), 69.75 (C-3), 69.24 (C-5′), 69.09 (C-5), 68.41 (C-3′), 66.31(C-4′), 66.09 (C-4), 62.35(C-6a), 62.03(C-6′), 55.00 (OCH2C≡CH), 20.90, 20.87, 20.74, 20.71, 20.67, 20.67, 20.65 (7C, CH3COO).
HRMS: m/z = 697.1950 [M + Na+] (calculated 697.1950).
3.8. Enzymatic Deprotection of Disaccharides 10–18
The deacetylated disaccharides were produced following a general procedure of hydrolysis.
The substrates (10 mM final concentration; 5 mM for 12) were dissolved in acetonitrile (20%–30% v/v depending on substrate solubility) under magnetic stirring, and then phosphate buffer (50 mM, pH 4.0–5.8) was added slowly. The reaction was started through the addition of immobilized CRL and/or AXE, previously conditioned with reaction buffer. The reactions were performed at 25 °C under mechanical stirring; the pH of the solution was maintained constant by automatic titration. Reaction course was monitored by TLC.
After complete consumption of the starting substrate or before an excessive formation of undesired products, the reactions were stopped by enzymatic derivative filtration on Büchner funnel. Acetonitrile was evaporated under reduced pressure, and the solution was brined and extracted with ethyl acetate. The organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The mixture obtained was purified by flash chromatography.
2′,3′,4′,6′-Tetra-O-acetyl-α-d-mannopyranosyl-(1→6)-2,3,4-tri-O-acetyl-α-d-mannopyranose (10a)
2′,3′,4′,6′-Tetra
-O-acetyl-α
-d-mannopyranosyl-(1→6)-1,2,3,4-tetra
-O-acetyl-α
-d-mannopyranose (
10) was hydrolyzed to the corresponding 2′,3′,4′,6′-tetra
-O-acetyl-α
-d-mannopyranosyl-(1→6)-2,3,4-tri
-O-acetyl-α
-d-mannopyranose (
10a) following the general procedure for enzymatic hydrolysis: substrate (10 mg, 10 mM) was solubilized in 1.7 mL of phosphate buffer 50 mM pH 4.8 and 30%
v/
v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (52 UI). The reaction mixture was monitored by TLC (dichloromethane/methanol 9:1, Rf = 0.76). The product was identified by comparing the reaction mixture with standard material produced in-house [
53].
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside (11a)
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (11) was hydrolyzed to the corresponding cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside (11a) following the general procedure for enzymatic hydrolysis: substrate (69 mg, 10 mM) was solubilized in 8.5 mL of phosphate buffer 50 mM pH 5.3 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (560 UI). The reaction mixture was monitored by TLC (dichloromethane/acetone 8:2, Rf = 0.53). Column chromatography (SiO2, dichloromethane/acetone 8:2) gave the desired product as a white sticky solid (15 mg, 23%).
1H-NMR (400 MHz, CDCl3): δ 5.58 (d, 1H, J = 1.4 Hz, H-1), 5.36 (t, 1H, J = 9.9 Hz, H-4), 5.33-5.29 (m, 2H, H-4′, H-3′), 5.24 (t, 1H, J = 2.2 Hz, H-2′), 5.18 (dd, 1H, J = 9.9, 3.2 Hz, H-3), 4.90 (d, 1H, J = 1.7 Hz, H-1′), 4.33-4.24 (m, 2H, H-6′a, H-5), 4.22 (dd, 1H, J = 3.3, 1.4 Hz, H-2), 4.17 (dd, 1H, J = 12.2, 2.6 Hz, H-6′b), 4.13-4.07 (m, 1H, H-5′), 3.87 (dd, 1H, J = 10.8, 7.0 Hz, H-6a), 3.60 (dd, 1H, J = 10.7, 2.3 Hz, H-6b), 3.56 (d, 1H, J = 17.3 Hz, SCH2CN), 3.38 (d, 1H, J = 17.3 Hz, SCH2CN), 2.18, 2.14, 2.12, 2.08, 2.08, 2.02 (5s, 18H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 170.83, 170.22, 170.08, 169.44, 169.81, 169.80 (6C, COO), 115.88 (SCH2CN), 97.00 (C-1′), 83.33 (C-1), 71.69 (C-3), 70.37 (C-5), 69.53 (C-2, C-2′), 68.92 (C-5′), 68.72, 66.49 (C-4), 65.96 (C-6), 65.88, 62.53 (C-6′), 20.88, 20.80, 20.77, 20.71, 20.67, 20.67 (6C, CH3COO), 15.21 (SCH2CN).
MS: m/z = 672.09 [M + Na+] (calculated 672.16).
(4-Methylphenyl) (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside (12a)
(4-Methylphenyl) (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl-(1→6)-2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (12) was hydrolyzed to the corresponding (4-methylphenyl) (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside(12a) following the general procedure for enzymatic hydrolysis: substrate (75 mg, 10 mM) was solubilized in 20.37 mL of phosphate buffer 50 mM pH 5.4 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (420 UI). The reaction mixture was monitored by TLC (dichloromethane/acetone 9:1, Rf = 0.22). Column chromatography (SiO2, dichloromethane/acetone 9:1) gave the desired product as a white sticky solid (17 mg, 24%).
1H-NMR (400 MHz, CDCl3): δ 7.39 (d, 2H J = 8.0 Hz, Ar), 7.17 (d, 2H, J = 8.0 Hz, Ar), 5.46 (d, J = 1.9 Hz, 1H, H-1), 5.42 (d, 1H, J = 9.9 Hz, H-4) 5.36-5.24 (m, 4H, H-2′, H-3′, H-4′, H-3), 4.92 (d, 1H, J = 1.6 Hz, H-1′), 4.50 (ddd, 1H, J = 10.0, 5.0, 2.4 Hz, H-5), 4.35-4.31 (m, 1H, H-2) 4.28 (dd, 1H, J = 12.3, 5.1 Hz, H-6′a), 4.09 (dd, 1H, J = 12.3, 2.5 Hz, H-6′b), 4.03 (ddt, 1H, J = 7.5, 5.1, 2.2 Hz, 1H, H-5′), 3.85 (dd, 1H, J = 11.4, 5.0 Hz, H-6a), 3.62 (dd, 1H, J = 11.4, 2.5 Hz, H-6b), 2.34 (s, 3H, CH3-Ar), 2.16, 2.12, 2.11, 2.09, 2.07, 2.02 (6s, 18H, COCH3).
13C-NMR (400 MHz, CDCl3): δ 170.72, 170.17, 169.98, 169.76, 169.76, 169.74 (6C, CH3COO), 138.07, 132.21, 130.06, 129.39 (Ar), 97.81 (C-1′), 88.06 (C-1), 71.86 (C-3), 70.59 (C-2), 70.28 (C-5), 69.62, 69.00, 68.61 (C-5′), 66.84 (C-6), 66.69 (C-4), 66.05, 62.35 (C-6′), 21.13(CH3-Ar), 20.89, 20.89, 20.75, 20.73, 20.73, 20.67 (6C, CH3COO).
MS: m/z = 723.24 [M + Na+] (calculated 723.19).
Ethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside (13a)
Ethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-1-thio-α-d-mannopyranoside (13) was hydrolyzed to the corresponding ethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside (13a) following the general procedure for enzymatic hydrolysis: substrate (28 mg, 10 mM) was solubilized in 4.39 mL of phosphate buffer 50 mM pH 4.8 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (160 UI). The reaction mixture was monitored by TLC (dichloromethane/acetone 8:2, Rf = 0.62). Column chromatography (SiO2, dichloromethane/acetone 9:1) gave the desired product as a white solid (10 mg, 38%).
1H-NMR (400 MHz, CDCl3): δ 5.35 (m, 1H, H-1) 5.33-5.22 (m, 5H, H-4, H-4′, H-3′, H-2′,H-3,), 4.90 (d, 1H, J = 1.8 Hz, H-1′), 4.40 (ddd, 1H, J = 9.3, 6.5, 2.2 Hz, H-5), 4.26-4.14 (m, 3H, H-6′a, H-6′b, H-2) 4.11 (ddd, 1H, J = 9.7, 5.4, 2.4 Hz, 1H, H-5′), 3.83 (dd, 1H, J = 10.9, 6.6 Hz, H-6a), 3.57 (dd, 1H, J = 10.9, 2.2 Hz, H-6b), 2.75-2.59 (m, 2H, SCH2CH3), 2.17, 2.13, 2.10, 2.07, 2.06, 2.01 (6s, 18H, COCH3), 1.35 (t, 3H, J = 7.4, SCH2CH3).
13C-NMR (400 MHz, CDCl3): δ 170.82, 170.17, 169.94, 169.81, 169.81, 169.81 (6C, CH3COO), 97.39 (C-1′), 83.21 (C-1), 72.03, 70.64 (C-2), 69.61, 69.43 (C-5), 68.84, 68.53 (C-5′), 66.96, 66.47 (C-6), 66.15, 62.47 (C-6′), 24.79 (SCH2CH3), 20.90, 20.87, 20.78, 20.74, 20.70, 20.67 (6C, CH3COO), 14.56 (SCH2CH3).
MS: m/z = 661.22 [M + Na+] (calculated 661.18).
Propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-α-d-mannopyranoside (14a)
Propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-α-d-mannopyranoside (14) was hydrolyzed to the corresponding propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-α-d-mannopyranoside (14a) following the general procedure for enzymatic hydrolysis: substrate (81 mg, 10 mM) was solubilized in 12 mL of phosphate buffer 50 mM pH 4.0 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (404 UI). The reaction mixture was monitored by TLC (dichloromethane/acetone 8:2, Rf = 0.41) and purified by flash chromatography (SiO2, dichloromethane/acetone 8:2). The desired product was obtained as a as a colorless oil (18.4 mg, 24%).
1H-NMR (400 MHz, CDCl3): δ 5.38-5.25 (m, 5H, H-4′, H-3′, H-2′, H-4, H-3), 5.10 (d, 1H, J = 1.8 Hz, H-1), 4.92 (d, 1H, J = 1.8 Hz, H-1′), 4.31 (d, 2H, J = 2.4 Hz, OCH2C≡CH), 4.26 (dd, 1H, J = 5.5 Hz, 12.4 Hz, H-6′a), 4.19-4.10 (m, 2H, H-6′b, H-5′), 4.09 (dd, 1H, J = 2.9, 1.9 Hz, H-2), 4.00 (ddd, 1H, J = 8.9, 6.0, 2.3 Hz, H-5), 3.81 (dd, 1H, J = 5.9 Hz, 11 Hz, H-6b), 3.61 (dd, 1H, J = 2.4 Hz, 11 Hz, H-6a), 2.52 (t, 1H, J = 2.4 Hz, C≡CH), 2.17, 2.13, 2.11, 2.07, 2.06, 2.01 (6s, 18H, COCH3).
13C-NMR (400 MHz, CDCl3): δ 170.78, 170.16, 169.88, 169.88, 169.88, 169.81 (6C, CH3COO), 97.86 (C-1), 96.35 (C-1′), 78.32 (OCH2C≡CH), 75.36 (OCH2C≡CH), 71.48, 69.83 (H-5), 69.58, 69.18, 68.99 (C-2), 68.65 (C-5′), 66.67 (C-6), 66.60, 66.04, 62.46 (C-6′), 54.82 (OCH2C≡CH), 20.88, 20.88, 20.77, 20.72, 20.72, 20.68 (6C, CH3COO).
MS: m/z = 655.58 [M + Na+] (calculated 655.19).
Allyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-α-d-mannopyranoside (15a)
Allyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-α-d-mannopyranoside (15) was hydrolyzed to the corresponding Allyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-3,4-di-O-acetyl-α-d-mannopyranoside (15a) following the general procedure for enzymatic hydrolysis: substrate (103 mg, 10 mM) was solubilized in 15.2 mL of phosphate buffer 50 mM pH 4.8 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (504 UI). The reaction mixture was monitored by TLC (dichloromethane/acetone 8:2, Rf = 0.33). Column chromatography (SiO2, dichloromethane/acetone 8:2,) gave the desired product as a white solid (50 mg, 52%).
1H-NMR (400 MHz, 45 °C, CDCl3): δ 5.94 (dddd, 1H, J = 17.2, 10.4, 6.2, 5.3, CH2CH=CH2), 5.39-5.24 (m, 7H, H-3, H-4, H-2′, H-3′, H-4′, CH2CH=CH2), 4.91 (d, 1H, J = 1.8 Hz, H-1) 4.90 (d, 1H, J = 1.8 Hz, H-1′), 4.28-4.21 (m, 2H, H-6′a, CH2CH=CH2), 4.16 (dd, 1H, J = 7.9, 2.3 Hz, H-6′b), 4.15-4.03 (m, 3H, H-2, H-5′, CH2CH=CH2), 3.99 (ddd, 1H, J = 9.9, 6.1, 2.4 Hz, H-5), 3.80 (dd, 1H, J = 10.9, 6.2 Hz, H-6a), 3.58 (dd, 1H, J = 10.9, 2.3 Hz, H-6b), 2.17, 2.12, 2.10, 2.05, 2.06, 2.00 (6s, 18H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 170.79, 170.15, 169.94, 169.93, 169.83, 169.80 (6C, CH3COO), 133.25 (CH2CH=CH2), 118.23 (CH2CH=CH2), 98.19 (C-1), 97.25 (C-1′), 71.73, 69.62, 69.35, 69.29, 68.91, 68.60, 68.40, 66.76, 66.63, 66.11, 62.46 (11C, carbon ring), 20.90, 20.89, 20.77, 20.74. 20.70, 20.67 (6C, CH3COO).
MS: m/z = 657.25 [M + Na+] (calculated 657.20).
Cyanomethyl (3′,4′,6′-tri-O-acetyl-α-d-mannopyranosyl)-(1→6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-d-glucopyranoside (16a)
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-d-glucopyranoside (16) was hydrolyzed to the corresponding cyanomethyl (3′,4′,6′-tri-O-acetyl-α-d-mannopyranosyl)-(1→6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-1-thio-β-d-glucopyranoside (16a) following the general procedure for enzymatic hydrolysis: substrate (80.5 mg, 10 mM) was solubilized in 11.6 mL of phosphate buffer 50 mM pH 4.5 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (378 UI). The reaction mixture was monitored by TLC (dichloromethane/methanol 9:1, Rf = 0.42). Column chromatography (SiO2, dichloromethane/acetone 8:2) gave the desired product as a white sticky solid (15.4 mg 20.5%).
1H-NMR (400 MHz, CDCl3): δ 6.02 (d, 1H, J = 9.3 Hz, NH), 5.39–5.07 (m, 4H, H-4′, H-3′, H-3, H-4), 4.95 (d, 1H, J = 1.9 Hz, H-1′), 4.75 (d, 1H, J = 10.4 Hz, H-1), 4.29 (dd, 1H, J = 12.3, 4.9 Hz, H-6′a), 4.25-4.10 (m, 3H, H-2, H-2′, H-6′b), 3.98 (ddd, 1H, J = 9.9, 4.9, 2.4 Hz, H-5′) 3.85-3.64 (m, 4H, CHCN, H-6a, H-5, H-6b), 3.34 (d, 1H, J = 17.3 Hz, CHCN), 2.12, 2.10, 2.08, 2.07, 2.05, 1.99 (6s, 18H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 171.33, 170.84, 170.60, 170.01, 169.84, 169.26 (6C, CH3COO), 116.53 (SCH2CN), 99.63 (C-1′), 83.36 (C-1), 77. 18 (C-5), 73.46 (C-3), 71.35 (C-3′), 68.92 (C-5′), 68.78 (C-2′), 68.67 (C-4), 66.23 (C-6), 65.95 (C-4′), 62.48 (C-6′), 52.57 (C-2), 23.11 (CH3, NHAc), 20.90, 20.81, 20.4, 20.66, 20.64 (5C, CH3COO), 14.50 (SCH2CN).
MS: m/z = 671.21 [M + Na+] (calculated 671.17).
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside (17a)
Cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-1-thio-α-d-mannopyranoside (17) was hydrolyzed to the corresponding cyanomethyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4-di-O-acetyl-1-thio-α-d-mannopyranoside (17a) following the general procedure for enzymatic hydrolysis: substrate (95 mg, 10 mM) was solubilized in 13.10 mL of phosphate buffer 50 mM pH 5.0 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (477 UI). The reaction mixture was monitored by TLC (dichloromethane/acetone 8:2, Rf = 0.33). Column chromatography (SiO2, dichloromethane/acetone 8:2) gave the desired product as a white solid (14.5 mg, 16%).
1H-NMR (400 MHz, CDCl3): δ 5.55 (d, 1H, J = 1.8 Hz, H-1), 5.30 (dd, 1H, J = 9.9, 3.4 Hz, H-3′), 5.23 (t, 2H, J = 10.3 Hz, H-4, H-4′), 5.18 (dd, 1H, J = 3.4, 1.9, H-2′), 5.11 (dd, 1H, J = 9.7, 3.3, H-3), 4.86 (d, 1H, J = 1.9 Hz, H-1′), 4.22 (dd, 1H, H-6′a), 4.16-4.09 (m, 2H, H-2, H-5′), 4.06 (dd, 1H, J = 11.9, 2.9 Hz, H-6′b), 4.00 (m, broad, 1H, J = 10.1, 4.5, 2.4 Hz, H-5), 3.75-3.58 (m, 2H, H-6a, H-6b), 3.41 (d, 1H, J = 17.2 Hz, SCH2CN), 3.31 (d, 1H, J = 17.2 Hz, SCH2CN), 2.08, 2.05, 2.04, 2.02, 2.01, 1.95 (6s, 18H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 170.88, 170.36, 170.34, 169.91, 169.91, 169.67 (6C, CH3COO), 116.06 (SCH2CN), 99.06 (C-1′), 83.49 (C-1), 76.86 (C-2), 72.27 (C-5), 70.27 (C-3), 69.51, 69.41 (C-5′, C-2′), 68.46 (C-3′), 66.46, 65.87 (C-4, C-4′), 62.83 (C-6′), 61.07 (C-6), 20.86, 20.84, 20.74, 20.71, 20.64, 20.61 (6C, CH3COO), 15.02 (SCH2CN).
MS: m/z = 672.09 [M + Na+] (calculated 672.16).
Propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4-di-O-acetyl-α-d-mannopyranoside (18a)
Propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-α-d-mannopyranoside (18) was hydrolyzed to the corresponding propargyl (2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-(1→2)-3,4-di-O-acetyl-α-d-mannopyranoside (18a) following the general procedure for enzymatic hydrolysis: substrate (38 mg, 10 mM) was solubilized in 5.6 mL of phosphate buffer 50 mM pH 5.8 and 30% v/v of acetonitrile. The reaction was started through addition of AXE-GLX-AG (102 UI). The reaction mixture was monitored by TLC (dichloromethane/acetone 8:2). Column chromatography (SiO2, dichloromethane/acetone 9:1, Rf = 0.17) gave the desired product as a white solid (7 mg, 20%).
1H-NMR (400 MHz, CDCl3): δ 5.40 (dd, 1H, J = 10.0, 3.3 Hz, H-3′), 5.37-5.26 (m, 4H, H-3, H-4, H-4′, H-2′), 5.19 (d, 1H, J = 2.0 Hz, H-1), 4.95 (d, 1H, J = 1.9 Hz, H-1′), 4.29 (d, 2H, J = 2.4 Hz, OCH2C≡CH), 4.27-4.15 (m, 3H, H-5′, H-6′a, H-6′b,), 4.10 (dd, 1H, J = 3.2, 2.0 Hz, H-2), 3.78 (ddd, 1H, J = 9.6, 4.4, 2.3 Hz, H-5), 3-75-3.71 (m, 2H, H-6a, H-6b), 2.50 (t, 1H, J = 2.4 Hz, OCH2C≡CH), 2.17, 2.13, 2.11, 2.09, 2.08, 2.03 (6s, 18H, CH3COO).
13C-NMR (400 MHz, CDCl3): δ 170.91, 170.39, 170.35, 169.92, 169.89, 169.63 (6C, CH3COO), 99.09 (C-1′), 96.86 (C-1), 78.20 (OCH2C≡CH), 76.75 (C-2), 75.53 (OCH2C≡CH), 71.33 (C-5), 69.96, 69.64, 69.13 (C-5′), 68.60 (C-3′), 66.58, 66.29, 62.65 (C-6′), 61.28 (C-6), 54.89 (OCH2C≡CH), 20.87, 20.83, 20.73, 20.73, 20.73, 20.67, (6C, CH3COO).
MS: m/z = 655.20 [M + Na+] (calculated 655.19).